Aikaterini Alexaki

Cellular Reservoirs of HIV-1 and their Role in Viral Persistence

A major obstacle in human immunodeficiency virus type 1 (HIV-1) eradication is the ability of the virus to remain latent in a subpopulation of the cells it infects. Latently infected cells can escape the viral immune response and persist for long periods of time, despite the presence of successful highly active antiretroviral therapy (HAART). Given the appropriate stimulus, latently infected cells can reactivate and start producing infectious virions. The susceptibility of these cell populations to HIV-1, their life span, their proliferative capacity, and their ability to periodically produce infectious virus subsequent to alterations in cellular physiology and/or immunologic controls are critical issues which determine the contribution of these cells to viral persistence. Memory CD4+ T cells due to the long life span, which may be several years, and their ability to reactivate upon encounter with their cognate antigen or other stimulation, are considered a critical reservoir for maintenance of latent HIV-1 proviral DNA. Cells of the monocyte-macrophage lineage, which originate in the bone marrow (BM), are of particular importance in HIV-1 persistence due to their ability to cross the blood-brain barrier (BBB) and spread HIV-1 infection in the immunoprivileged central nervous system (CNS). Hematopoietic progenitor cells (HPCs) are also a potential HIV-1 reservoir, as several studies have shown that CD34+ HPCs carrying proviral DNA can be found in vivo in a subpopulation of HIV-1-infected patients. The ability of HPCs to proliferate and potentially generate clonal populations of infected cells of the monocyte-macrophage lineage may be crucial in HIV-1 dissemination. The contribution of these and other cell populations in HIV-1 persistence, as well as the possible strategies to eliminate latently infected cells are critically examined in this review.

HIV-1 Infection of Bone Marrow Hematopoietic Progenitor Cells and Their Role in Trafficking and Viral Dissemination

Patients with HIV-1 often present with a wide range of hematopoietic abnormalities, some of which may be due to the presence of opportunistic infections and to therapeutic drug treatments. However, many of these abnormalities are directly related to HIV-1 replication in the bone marrow (BM). Although the most primitive hematopoietic progenitor cells (HPCs) are resistant to HIV-1 infection, once these cells begin to differentiate and become committed HPCs they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and infectious virus production. Trafficking of BM-derived HIV-1-infected monocytes has been shown to be involved in the dissemination of HIV-1 into the central nervous system (CNS), and it is possible that HIV-1 replication in the BM and infection of BM HPCs may be involved in the early steps leading to the development of HIV-1-associated dementia (HAD) as an end result of this cellular trafficking process. In addition, the growth and development of HPCs in the BM of patients with HIV-1 has also been shown to be impaired due to the presence of HIV-1 proteins and changes in the cytokine milieu, potentially leading to an altered maturation process and to increased cell death within one or more BM cell lineages. Changes in the growth and differentiation process of HPCs may be involved in the generation of monocyte populations that are more susceptible and/or permissive to HIV-1, and have potentially altered trafficking profiles to several organs, including the CNS. A monocyte subpopulation with these features has been shown to expand during the course of HIV-1 disease, particularly in HAD patients, and is characterized by low CD14 expression and the presence of cell surface CD16.

AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation

Human T cell leukemia virus type 1 (HTLV‐1) has previously been shown to infect antigen‐presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV‐1‐associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV‐1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV‐1 viral gene expression in established monocytic cell lines was examined. U‐937 promonocytic cells transiently transfected with a HTLV‐1 long‐terminal repeat (LTR) luciferase construct were treated with phorbol 12‐myristate 13‐acetate (PMA) to induce cellular differentiation. PMA‐induced cellular differentiation resulted in activation of basal and Tax‐mediated transactivation of the HTLV‐1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA‐induced cellular differentiation induced DNA‐binding activity of cellular transcription factors to Tax‐responsive element 1 (TRE‐1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP‐1) family of basic region/leucine zipper proteins (Fra‐1, Fra‐2, JunB, and JunD) were induced to bind to TRE‐1 repeat II during cellular differentiation. Inhibition of AP‐1 DNA‐binding activity by overexpression of a dominant‐negative c‐Fos mutant (A‐Fos) in transient expression analyses resulted in severely decreased levels of HTLV‐1 LTR activation in PMA‐induced U‐937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV‐1 viral gene expression may become up‐regulated by AP‐1 during differentiation into macrophages or dendritic cells.

Impact of Viral Activators and Epigenetic Regulators on HIV-1 LTRs Containing Naturally Occurring Single Nucleotide Polymorphisms

Following human immunodeficiency virus type 1 (HIV-1) integration into host cell DNA, the viral promoter can become transcriptionally silent in the absence of appropriate signals and factors. HIV-1 gene expression is dependent on regulatory elements contained within the long terminal repeat (LTR) that drive the synthesis of viral RNAs and proteins through interaction with multiple host and viral factors. Previous studies identified single nucleotide polymorphisms (SNPs) within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3T, C-to-T change at position 3, and 5T, C-to-T change at position 5 of the binding site, respectively, when compared to the consensus B sequence) that are low affinity binding sites and correlate with more advanced stages of HIV-1 disease. Stably transfected cell lines containing the wild type, 3T, 5T, and 3T5T LTRs were developed utilizing bone marrow progenitor, T, and monocytic cell lines to explore the LTR phenotypes associated with these genotypic changes from an integrated chromatin-based microenvironment. Results suggest that in nonexpressing cell clones LTR-driven gene expression occurs in a SNP-specific manner in response to LTR activation or treatment with trichostatin A treatment, indicating a possible cell type and SNP-specific mechanism behind the epigenetic control of LTR activation.

Effect of μ-opioid agonist DAMGO on surface CXCR4 and HIV-1 replication in TF-1 human bone marrow progenitor cells.

BackgroundApproximately one-third of the AIDS cases in the United States have been attributed to the use of injected drugs, frequently involving the abuse of opioids. Consequently, it is critical to address whether opioid use directly contributes to altered susceptibility to HIV-1 beyond the increased risk of exposure. Previous in vitro and in vivo studies addressing the role of μ-opioid agonists in altering levels of the co-receptor CXCR4 and subsequent HIV-1 replication have yielded contrasting results. The bone marrow is believed to be a potential anatomical sanctuary for HIV-1.MethodsThe well-characterized CD34+CD38+ human bone marrow–derived hematopoietic progenitor cell line TF-1 was used as a model to investigate the effects of the μ-opioid receptor–specific peptide DAMGO (D-Ala2,N-Me-Phe4, Gly5-ol-enkephalin) on CXCR4 expression as well as infection of undifferentiated human hematopoietic progenitor cells.ResultsThe results revealed the presence of the μ-opioid receptor-1 isoform (MOR-1) on the surface of TF-1 cells. Furthermore, immunostaining revealed that the majority of TF-1 cells co-express MOR-1 and CXCR4, and a subpopulation of these double-positive cells express the two receptors in overlapping membrane domains. Three subpopulations of TF-1 cells were categorized based on their levels of surface CXCR4 expression, defined as non-, low-, and high-expressing. Flow cytometry indicated that treatment with DAMGO resulted in a shift in the relative proportion of CXCR4+ cells to the low-expressing phenotype. This result correlated with a >3-fold reduction in replication of the X4 HIV-1 strain IIIB, indicating a role for the CXCR4 high-expression subpopulation in sustaining infection within this progenitor cell line.ConclusionsThese experiments provide insight into the impact of μ-opioid exposure with respect to inhibition of viral replication in this human TF-1 bone marrow progenitor cell line model.

Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site.

Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.

Development of a Human Bone Marrow Progenitor Cell Line to Examine HIV-1 Susceptibility and LTR Activity

Previous studies have suggested that the bone marrow compartment may play an integral role in the genesis of HIV-1 dementia (HIVD). Interestingly, CD34+/CD38pluripotent stem cells within the bone marrow are refractile to HIV-1 infection. The CD34+/CD38+ TF-1 cell line has been selected as a model to study HIV-1 infection during the differentiation process of hematopoietic progenitor cells. A number of cytokines such as GM-CSF, M-CSF, IL-1β, TNF-α, and IL-4 were used to induce differentiation and activation of TF-1 cells and their surface marker expression was monitored by flow cytometry. Interestingly, IL-1β treatment, alone or in combination with TNFα, lead to up-regulation of CXCR4 and CCR5 surface presentation, and preservation of CD4 expression possibly providing an optimal cellular phenotype for HIV-1 infection of this cell population. The surface marker expression after this treatment also correlated with a more differentiated phenotype. To begin exploring the potential of these cells to support productive HIV-1 replication, a series of stably transfected cell lines were developed. To this end, macrophage-, T celland dual-tropic long terminal repeats (LTRs) were coupled to the gene encoding green fluorescent protein. These cell lines were utilized to explore the functional properties of specific cis-acting regulatory elements in LTR function within the bone marrow precursor cell population. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Characterization of C/EBP Binding Sites Downstream of the Transcriptional Start Site in the HIV-1 LTR

Previous studies have shown that at least one upstream CCAAT enhancer binding protein (C/EBP) site was necessary for HIV-1 LTR activity in cells of the monocyte/macrophage lineage. However, no investigation has been performed to date on C/EBP sites downstream (DS) of the start of transcription. Analyses of 115 clade B LTRs indicated there are three potential C/EBP sites within the downstream LTR region. Electrophoretic mobility shift (EMS) analyses demonstrated one of the three sites (DS3) was able to bind members of the C/EBP family. Analyses of clade A, C, and D LTRs indicated this site was highly conserved among different clades, suggesting the presence of a functionally important cis-acting element. In comparison to the clade B consensus DS3 element, EMS analysis demonstrated the DS3 7A variant exhibited a relative high affinity for C/EBP factors, while the 3C and 7G configurations exhibited lower affinities. Additional studies demonstrated specific DS3 variants exhibited differences in relative affinity for full-length and truncated C/EBPb. Transient transfection studies utilizing parental LTRs derived from LAI, YU.2, and 89.6 molecular clones containing the DS3 7A, 3C, or 7G variants exhibited altered LTR activity compared to their parental strains. These results have suggested that DS3 plays a role in regulating HIV-1 transcription. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Development of a Quantum Dot-based Assay System for Detection of Specific HIV-1 LTR Sequence Variants

Analysis of human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR) sequence variation within the CCAAT/enhancer binding protein (C/EBP) and stimulating protein (Sp) transcription factor binding sites has identified variants that correlate with HIV-associated dementia (HIVD). CdSe/ZnS nanocrytsals have facilitated the investigation of nanoand peco-scale biological components. Quantum dot-conjugated oligonucleotides homologous to specific variants of Sp site III and C/EBP site I, were utilized to quantitate the relative abundance of specific LTR variants. Quantum dot-conjugated oligonucleotides containing the Sp site III 5T binding site variant (C-to-T change at position 5) or the C/EBP site I 3T site variant, were reacted with plasmid DNA containing increasing concentrations of plasmid with the homologous LTR sequence variant. The results suggest that quantum dot-conjugated oligonucleotides specific for sequence variants within the LTR can be used as reporter molecules for identification and quantitation of HIV-1 genetic variation. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005